The Candida albicans phosphatase Inp51p interacts with the EH domain protein Irs4p, regulates phosphatidylinositol-4,5-bisphosphate levels and influences hyphal formation, the cell integrity pathway and virulence.
We previously identified Candida albicans Irs4p as an epidermal growth factor substrate 15 homology (EH) domain-containing protein that is reactive with antibodies in the sera of patients with candidiasis and contributes to cell wall integrity, hyphal formation and virulence. In this study, we use a yeast two-hybrid method and co-immunoprecipitation to show that Irs4p physically interacts with the phosphatase Inp51p. Disruption of the Inp51p Asn-Pro-Phe (NPF) motif eliminates the interaction, suggesting that this motif is targeted by Irs4p. Both inp51 and irs4 null mutants exhibit significantly increased levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)] without changes in levels of other phosphoinositides. Like the irs4 mutant, the inp51 mutant demonstrates increased susceptibility to cell wall-active agents, impaired hyphal formation and abnormal chitin distribution along hyphal walls during growth within solid agar. Moreover, the inp51 and irs4 mutants overactivate the cell wall integrity pathway as measured by Mkc1p phosphorylation. As anticipated, mortality due to disseminated candidiasis is significantly attenuated among mice infected with the inp51 mutant, and tissue burdens and inflammation within the kidneys are reduced. Hyphal formation and chitin distribution in vivo are also impaired, consistent with observations of embedded growth in vitro. All phenotypes exhibited by the inp51 and irs4 mutants are rescued by complementation with the respective genes. In conclusion, our findings suggest that Irs4p binds and activates Inp51p to negatively regulate PI(4,5)P(2) levels and the cell integrity pathway, and that PI(4,5)P(2) homeostasis is important for coordinating cell wall integrity, hyphal growth and virulence under conditions of cell wall stress.
Badrane
Hassan
H
Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA.
Nguyen
M Hong
MH
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.|North Florida/South Georgia Veterans Health System, Gainesville, FL, USA.|Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA.
Cheng
Shaoji
S
Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA.
Kumar
Vipul
V
Department of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, FL, USA.
Derendorf
Hartmut
H
Department of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, FL, USA.
Iczkowski
Kenneth A
KA
Department of Pathology, University of Colorado Health Science Center, Aurora, CO, USA.
Clancy
Cornelius J
CJ
North Florida/South Georgia Veterans Health System, Gainesville, FL, USA.|Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA.
D000818:Animals; D002176:Candida albicans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity*; D002177:Candidiasis / Q000382:microbiology*; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D025301:Hyphae / Q000737:chemistry / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014774:Virulence; D000099182:Phosphoinositide 5-Phosphatases
D005656:Fungal Proteins; D055504:Insulin Receptor Substrate Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010744:Phosphoric Monoester Hydrolases; D000099182:Phosphoinositide 5-Phosphatases